Vesicular acetylcholine transporter (VAChT) stores acetylcholine (ACh) in synaptic vesicles for evoked [unreadable]elease from cholinergic nerve terminals. It is allosterically inhibited by a compound called vesamicol. The project seeks understanding of VAChT structure and transport mechanism with the long term goal of developing a pharmacology to control ACh storage (and thus release) for treatment of cholinergic disorders. Experiments using site-directed mutagenesis, expression and characterization of mutant properties are proposed. A residue in VAChT must be protonated for transport. The first aim is to test whether the recent Dreliminary assignment of the aspartate residue in putative transmembrane domain XI (TMD XI) to this role s correct. A different aspartate or glutamate residue that recently was shown not to be in a TMD must be deprotonated to bind ACh and vesamicol. The second aim is to identify it. The third aim is to test and refine the recent assignments of ACh and vesamicol binding sites to putative TMDs XIII and X. The aspartate residue in putative TMD X is known to participate in proton translocation reactions that provide energy input to VAChT. The fourth aim is to test and refine the proposed role(s) of this residue using a novel assay that monitors transmembrane reorientation of the ACh binding site. The fifth aim is to test whether VAChT contains 12 TMDs, as is now assumed, by inserting one cysteine residue into each hydrophilic region of the sequence and determining which side of the membrane the residue is exposed to using a chemical labeling technique. The sixth aim is to identify which hydrophilic regions change conformation when VAChT binds ACh and vesamicol and transports ACh by using the labeling technique under conditions that detect different rates of labeling. The experiments will be extended to mutants of residues responsible for the required protonation and deprotonation to identify which hydrophilic regions change conformation at pH extremes. The results will provide substantial new information about VAChT structure and dynamics.